This invention relates to sequencing of DNA.
The sequence of nucleotide bases in DNA is generally determined using the methods described by Maxam and Gilbert (65 Methods Enzymol. 497, 1980) or by Sanger et al. (74 Proc. Natl. Acad. Sci. USA 5463, 1977). These methods generally involve the isolation of purified fragments of DNA prior to sequence determination.
Church et al. (81 Proc. Natl. Acad. Sci. 1991, 1984) describe a method of sequencing directly from genomic DNA. Unlabelled DNA fragments are separated in a denaturing gel after complete restriction endonuclease digestion and partial chemical cleavage of the genome. After binding these fragments to a nylon membrane the DNA is hybridized with a probe comprising RNA homologous to a region near to the region to be sequenced. The membrane can be reprobed with other probes to sequence other regions of interest.